The present invention relates to nucleic acid sequences encoding antigenic proteins associated with Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii; collectively designated Bb hereinafter), particularly polypeptides associated with virulence; vaccine formulations comprising these polypeptides are also part of the invention. The invention also relates to methods for producing Bb immunogenic polypeptides and corresponding antibodies. Other embodiments of the invention relate to methods for detecting Lyme disease and transformed cells comprising Bb associated nucleic acids.
Lyme disease is a multisystem disease resulting from tick transmission of the infectious agent, Bb (Rahn and Malawista, 1991). Although recognised as a clinical entity within the last few decades (Steere et al., 1977), case reports resembling Lyme disease date back to the early part of the 20th century. Cases of the disease have been reported in Europe, Asia and North America (Schmid, 1985). Despite a relatively low total incidence compared to other infectious diseases, Lyme disease represents a significant health problem because of its potentially severe cardiovascular, neurologic and arthritic complications, difficulty in diagnosis and treatment and high prevalence in some geographic regions.
Bb is not a homogeneous group but has a variable genetic content, which may in turn affect its virulence, pattern of pathogenesis and immunogenicity. Lyme borreliosis associated borreliae are so far taxonomically placed into three species, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (Burgdorfer et al., 1983, Baranton et al., 1992, Canica et al., 1993). It is well documented that considerable genetic, antigenic and immunogenic heterogeneity occurs among them, as well as among the strains within the separate species (Baranton et al., 1992, Canica et al., 1993, Zingg et al., 1993, Wilske et al., 1993, Adam et al., 1991, Marconi and Garon 1992). The major evidence of this phenomenon is provided by the molecular studies of the plasmid-encoded outer surface protein A (OspA), OspB and OspC (Barbour et al., 1984, Jonsson et al., 1992, Wilske et al., 1993, Marconi et al., 1993). In different animal models efficient protection is achieved by passive and active immunisation with OspA (Schaible et al., 1990, Fikrig et al., 1992, Erdile et al., 1993), and therefore, OspA remains one of the main candidates for Borrelia vaccine. It is unclear, however, whether inter- and intra-species heterogeneity of OspA, as well as other competitors for immunoprophylaxis, allow efficient cross-protection (Fikrig et al., 1992, Norris et al., 1992). Furthermore, it was recently suggested that certain protective antibodies produced early in the course of Borrelia infection are unrelated to OspA (Norton Hughes et al., 1993, Barthold and Bockenstedt, 1993).
Its virulence factors, pathogenetic mechanisms and means of immune evasion are unknown. At the level of patient care, diagnosis of the disease is complicated by its varied clinical presentation and the lack of practical, standardised diagnostic tests of high sensitivity and specificity. Antimicrobial therapy is not always effective, particularly in the later stages of the disease.
Variation among Bb strains and species and the changes resulting from in vitro passage add to the problems of developing vaccines or immunodiagnostics from either the whole organism or specifically associated proteins. Using a PCR assay, it was found that one set of oligonucleotide primers was specific for North American Bb isolates, another for most European isolates and a third set recognised all Bb strains (Rosa et al., 1989).
Serological assays for the diagnosis and detection of Lyme disease are thought to offer the best promise for sensitive and specific diagnosis. However, serologic assays generally use whole Bb as antigen and suffer from a low xe2x80x9csignal to noisexe2x80x9d ratio, i.e., a low degree of reactivity in positive samples, particularly early in the disease, as compared to negative samples. This problem results in high numbers of false negatives and the potential for false positives. Background reactivity in negative controls may be due in part to conserved antigens such as the 41K flagellin and the 60K xe2x80x9cCommon Antigenxe2x80x9d. These Bb proteins possess a high degree of sequence homology with similar proteins found in other bacteria. Therefore normal individuals will often express anti-flagellar and anti-60K antibodies. Unique, highly reactive Bb antigens for serological assays are therefore desirable but heretofore unavailable.
Diagnosis of Lyme disease remains a complex and uncertain endeavour, due to lack of any single diagnostic tool that is both sensitive and specific. Clinical manifestations and history are the most common bases for diagnosis. However, there is a pressing need for specific, sensitive, reproducible and readily available confirmatory tests. Direct detection offers proof of infection but is hampered by the extremely low levels of Bb that are typically present during infection, as well as the inaccessibility of sites that tend to be consistently positive (e.g., heart and bladder). Culture, although sensitive, is cumbersome and requires 1-3 weeks to obtain a positive result. PCR appears to offer promise in terms of direct detection (Lebech et al., 1991) and indeed Goodman et al. (1991) have reported detection of Bb DNA in the urine of patients with active Lyme disease using a PCR method. However, it is unlikely that PCR assays will become commonly used in clinical laboratories because of the degree of skill required for its use and the high risk of DNA contamination.
Another problem in detection of Lyme disease is the substantial number of humans exposed to Bb who develop inapparent or asymptomatic infections. This number has been estimated to be as high as 50% (Steere et al., 1986).
There is clearly a need for means of preparing Bb-specific antigens, e.g., for the development of diagnostic tests for Lyme disease. Adequate assays do not exist and should ideally meet several criteria, including (1) expression of an antigen by all pathogenic Bb strains, (2) elicitation of an immune response in all Lyme disease patients, (3) high immunogenicity with a detectable antibody response early in the infection stage, (4) antigens unique to Bb without cross-reactivity to other antigens, and (5) distinction between individuals exposed to non-pathogenic as opposed to pathogenic forms of Bb.
There have been several studies describing low molecular weight proteins from Bb. Katona et al. showed the presence of a major low-molecular weight lipoprotein specific for B. burgdorferi and raised the possibility that it was a borrelial equivalent of Braun""s lipoprotein (Katona et al., 1992). Another study reported an immunogenic 14 kDa surface protein of B. burgdorferi recognised by sera from Lyme disease patients (Sambri et al., 1991). A 14 kDa mitogenic lipoprotein of B. burgdorferi was reported by Honarvar et al. (1994).
Sadziene et al. (1994) when analysing an Osp-less B. burgdorferi strain identified a 13 kDa surface exposed protein which was designated p13.
It is an object of the invention to provide novel nucleic acid fragments and polypeptide fragments which are useful in the preparation of diagnostics and prophylactic means and compositions relating to infections with Bb. It is a further object to provide novel vaccines and diagnostic means as well as methods for the preparation and use of such vaccines and diagnostic means. Finally, it is an object of the invention to provide tools such as vectors and transformed cells which facilitate the preparation of the polypeptide fragments and the vaccines.
The inventors have surprisingly found that an antigen from Bb with an apparent molecular weight of 13 kDa (determined by SDS-PAGE, and subsequent visualization such as staining with Coomassie Blue) is highly conserved in the three strains B. burgdorferi sensu stricto B31, B. garinii IP90, and B. afzelii ACAI, whereas this antigen cannot be found in Borrelia species related to relapsing fever and avian borreliosis. The disclosed antigens are therefore excellent candidates for vaccines and diagnostics relating to infections with Bb. The antigens will be termed P13.
The present invention thus addresses one or more of the foregoing or other problems associated with the preparation and use of Bb specific antigens, particularly those antigens associated with virulence and which are useful for developing detection and diagnostic methods for Lyme disease. The present invention involves the identification of such antigens, which herein are designated P13 as well as the identification and isolation of Bb nucleic acid sequences that encode P13 antigens or antigenic polypeptides derived therefrom. These sequences are useful for preparing expression vectors for transforming host cells to produce recombinant antigenic polypeptides. It is further proposed that these antigens will be useful as vaccines or immunodiagnostic agents for Bb associated diseases such as Lyme disease in particular.
The DNA of the present invention was isolated from Bb. The microorganism is a spiral-shaped organism approximately 0.2 micron in diameter and ranging in length from about 10 to 30 microns. Like other spirochaetes, it possesses an inner membrane, a thin peptidoglycan layer, an outer membrane, and periplasmic flagella which lie between the inner and outer membranes. Bb is an obligate parasite found only in association with infected animals and arthropod vectors in endemic areas. Bb-like organisms have also been identified in birds raising the possibility that birds could also serve as an animal reservoir. While some Bb isolates have been cloned, most isolates have not been cloned and most likely represent mixtures of different variants even at the time of culture origin.
Bb has similarities with other relapsing fever organisms such as B. hermsii. Bb has a single chromosome with two unusual features, linear conformation and small size (approximately 900 kilobase pairs). Fresh isolates of Bb contain up to four linear plasmids and six circular supercoiled plasmids. The plasmid content of different Bb isolates is highly variable. For example, in one study only two of thirteen strains had similar plasmid profiles. Some plasmids are lost during in vitro passage which may correlate with loss of virulence. Outer surface proteins OspA and OspB are encoded on the 49 kbp linear plasmid. The P13 membrane-associated/outer surface proteins discovered by the inventors are encoded on the Bb chromosome. The P13 protein gene being localised to the chromosome of borreliae shows a higher degree of conservation among Lyme disease associated borreliae contrary to the plasmid-encoded major outer surface proteins A, B, and C which exhibit a significant species and strain dependent genetic and antigenic polymorphism (Barbour 1986, Jonsson et al., 1992, Wilske et al., 1993). Furthermore, the level of similarity and identity between the deduced amino acid sequence of the P13 protein from different borrelia strains further shows that this protein can be useful as a vaccine against Lyme disease as well as a target for diagnostic use.
In order to identify DNA segments encoding the P13 proteins, purified protein was isolated from B. burgdorferi B313 by preparative SDS-PAGE for subsequent use in amino acid sequencing. Attempts to N-terminally sequence the purified protein by standard techniques were unsuccessful. The protein was therefore subjected to V8 protease cleavage. After protease cleavage the peptide was transferred to polyvinylene diffusable membranes, and sequence analysis was performed using standard sequencing techniques (Matsudaira, 1987). A 25 amino acid sequence was identified (SEQ ID NO: 1).
DNA libraries were prepared by restriction enzyme digestion of DNA prepared from the strains B. burgdorferi B31, B. afzelii ACAI and B. garinii IP90.
Codons for the amino acid sequence obtained, SEQ ID NO: 1, were selected by reverse translation based on (1) conclusion that codons containing A or T were favoured and (2) knowledge of published DNA sequences for several Bb proteins. A choice favouring A or T containing codons was based on the observation that the G+C content of Bb is only 28-35% (Burman et al., 1990). Two oligonucleotides were synthesized having the sequences shown in in SEQ ID NO: 2 and SEQ ID NO:3. These were used as primers in a PCR reaction with DNA prepared from B. burgdorferi B31 as template. The amplified fragment was sequenced, SEQ ID NO: 4, and verified to code for the amino acid sequence, SEQ ID NO: 1.
A DNA probe, designated Y7 (SEQ ID NO:7), was designed and used to screen the DNA library prepared from B. burgdorferi B31 in an attempt to identify DNA encoding the P13 protein from this Bb species. This attempt proved unsuccessful.
An RsaI restriction site identified in the DNA sequence of the PCR fragment was used in a further attempt to clone the P13 gene. Bb DNA was digested with RsaI and the fragments cloned into a pUC plasmid. Further PCR amplification using the sequence identified surrounding the RsaI site yielded DNA fragments which were found to code for the P13 protein.
The identified sequence of the P13 gene from B. burgdorferi B31 was used to design PCR primers which were subsequently used to clone the P13 gene from B. afzelii ACAI and B. garinii IP90.
The P13 protein which has been cloned by the inventors of the present invention has been shown to have a molecular weight of about 19,000 as calculated from the deduced amino acid sequence of the full-length protein but a molecular weight of about 14,000 as determined by MS but nevertheless to be identical to a protein from Bb which has an apparent molecular weight in SDS-PAGE of 13 kDa. This difference can be explained by post-translational modifications of the P13 protein. This is in accordance with the observation that it was not possible by standard methods to obtain an N-terminal amino acid sequence of P13 protein prepared from Bb.
The deduced amino acid sequences of P13 from B. burgdorferi B31, B. afzelii ACAI and B. garinii IP90 were analysed and it was found that the N-terminal regions of the deduced amino acid sequences are typical of the signal peptides of bacterial proteins. These leader peptide sequences are typical of exported proteins with a basic residue followed by a hydrophobic and a potential leader peptidase I cleavage site according to the criteria established by von Heijne (1986).
Amino acid sequences resembling the signal sequences of bacterial lipoproteins can also be found in the N-terminal region of the deduced amino acid sequences. The N-terminal methionine is followed by a hydrophobic region and a signal peptidase 11 recognition sequence. The signal sequences, Leu Ala Thr Phe Cys for B. burgdorferi B31, Leu Leu Ala Phe Cys for B. afzelii ACAI and Leu Val lie Phe Cys for B. garinii I P90, differed somewhat from the consensus signal peptidase II recognition sequence (Leu Xaa Xaa Cys) found in most bacteria, but resembled the cleavage sequence Leu Ser Ile Ser Cys of the outer surface protein D (OspD) of Bb and Leu Met Ile Gly Cys of the variable major proteins Vmp7 and Vmp21 of B. hermsii. These surface antigens have been shown to be lipoproteins (Norris et al., 1992; Burman et al., 1990). The presence of this leader sequence may imply that mature P13 proteins are translocated across the cytoplasmic membrane and are anchored to the cytoplasmic membrane and/or outer membranes via fatty acids associated with an N-terminal cysteinyl residue. Lipidated forms of the outer surface protein A (OspA) from Bb have been shown to be more immunogenic that non-lipidated forms of OspA (Erdile et al., 1993).
However, it should be understood that when the terms xe2x80x9c13 kDa proteinxe2x80x9d or xe2x80x9c13 kDa antigenxe2x80x9d or xe2x80x9c13 kDa polypeptidexe2x80x9d are used in the present specification and claims, this is an alternative designation of the P13 polypeptide.
Antigenicity of the P13 protein was verified by immunisation of a rabbit. Antiserum collected from rabbits injected with the P13 protein prepared from B. burgdorferi B313 was found to recognise the P13 protein of B. burgdorferi B31, B. afzelii ACAI, and B. grainii IP90. There was no apparent reactivity of the antiserum with B. hemsii, B. crocidurae, B. anserina. 
The nucleic acid segments of the present invention encode antigenic amino acid sequences associated with Bb. These sequences are important for their ability to selectively hybridise with complementary stretches of Bb gene segments. Varying conditions of hybridization may be desired, depending on the application envisioned and the selectivity of the probe toward the target sequence. Where a high degree of selectivity is desired, one may employ relatively stringent conditions to form the hybrids, such as relatively low salt and/or high temperature conditions. Under these conditions, little mismatch between the probe and template or target strand is tolerated. Less stringent conditions might be employed when, for example, one desires to prepare mutants or to detect mutants when significant divergence exists.
In clinical diagnostic embodiments, nucleic acid segments of the present invention may be used in combination with an appropriate means, such as a label, to determine hybridization with DNA of a pathogenic organism. Typical methods of detection might utilise, for example, radioactive species, enzyme-active or other marker ligands such as avidin/biotin, which are detectable directly or indirectly. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as alkaline phosphatase or peroxidase rather than radioactive or other reagents that may have undesirable environmental effects. Enzyme tags, for example, often utilise calorimetric indicator substrates that are readily detectable spectrophotometrically, many in the visible wavelength range. Luminescent substrates could also be used for increased sensitivity.
Hybridisable DNA segments may include any of a number of segments of the disclosed DNA. For example, relatively short segments of at least 12 or so base pairs may be employed, or, more preferably when probes are desired, longer segments of at least 20, at least 30, and at least 40 base pairs, depending on the particular applications desired. Shorter segments are preferred as primers in molecular amplification techniques such as PCR, while some of the longer segments are generally preferable for blot hybridizations. It should be pointed out, however, that while sequences disclosed for the DNA segments of the present invention are defined by SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22, a certain amount of variation or base substitution would be expected, e.g., as may be found in mutants or strain variants, but which do not significantly affect hybridization characteristics. Such variations, including base modifications occurring naturally or otherwise, are intended to be included within the scope of the present invention.
While the Bb P13 antigens of the present invention have been disclosed in terms of the specific amino acid sequences SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, it is nonetheless contemplated that the amino acid sequences will be found to vary from species to species and from isolate to isolate. Moreover, it is quite clear that changes may be made in the underlying amino acid sequence through, e.g., site-directed mutagenesis of the DNA coding sequence, in a way that will not negate its antigenic capability.
The invention also relates to at least partially purified antigenic Bb proteins or polypeptides which are capable of producing an in vivo immunogenic response when challenged with Bb. These proteins may comprise all or part of the amino acid sequence encoded by the DNA disclosed herein. Particularly preferred antigenic proteins have the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23. Post-translationally modified forms of these antigenic proteins are also the subject of this invention. These proteins as well as their epitopes will be useful in connection with vaccine development, and as antigen(s) in immunoassays for detection of Bb antibodies in biological fluids such as serum, seminal or vaginal fluids, urine, saliva, body exudates and the like.
In other aspects, the invention concerns recombinant vectors such as plasmids, phages or viruses, which comprise DNA segments in accordance with the invention, for use in replicating such sequences or even for the expression of encoded antigenic peptides or proteins. Vectors or plasmids may be used to transform a selected host cell. In preparing a suitable vector for transforming a cell, desired DNA segments from any of several Bb sources may be used, including genomic fragments, cDNA or synthetic DNA. In the practice of the present invention, an expression vector may incorporate at least part of the DNA sequence of SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22, encoding one or more epitopic segments of the disclosed antigens of the present invention.
Expression vectors may be constructed to include any of the DNA segments hereinabove disclosed. Such DNA might encode an antigenic protein specific for virulent strains of Bb or even hybridization probes for detecting Bb nucleic acids in samples. Longer or shorter DNA segments could be used, depending on the antigenic protein desired. Epitopic regions of the disclosed proteins of the present invention expressed or encoded by the disclosed DNA could be included as relatively short segments of DNA. A wide variety of expression vectors are possible including, for example, DNA segments encoding reporter gene products useful for identification of heterologous gene products and/or resistance genes such as antibiotic resistance genes which may be useful in identifying transformed cells.
Recombinant vectors such as those described are particularly preferred for transforming bacterial host cells. Accordingly, a method is disclosed for preparing transformed bacterial host cells that generally includes the steps of selecting a suitable bacterial host cell, preparing a vector containing a desired DNA segment and transforming the selected bacterial host cell. Several types of bacterial host cells may be employed, including Bb, E. coli, B. subtilis, and the like as well as prokaryotic host cells.
Transformed cells may be selected using various techniques, including screening by differential hybridization, differential display techniques, identification of fused reporter gene products, resistance markers, anti-antigen antibodies and the like. After identification of an appropriate clone, it may be selected and cultivated under conditions appropriate to the circumstances, as for example, conditions favouring expression or, when DNA is desired, replication conditions.
Another aspect of the invention involves the preparation of antibodies and vaccines from the antigenic P13 proteins or epitopic regions of these proteins encoded by the disclosed DNA. It is expected that the sensitivity and specificity of an antibody response to these P13 proteins and their epitopes will be superior to the response that has been obtained from other Bb antigens that are not associated with virulence. Previous work with several Bb antigens isolated from both virulent and avirulent strains indicated low sensitivity when immunofluorescence and ELISA assays were employed, especially during early stages of infection.
In both immunodiagnostics and vaccine preparation, it is often possible and indeed more practical to prepare antigens from segments of a known immunogenic protein or polypeptide. Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide. Potential antigenic or immunogenic regions may be identified by any of a number of approaches, e.g., Jameson-Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods (1981) hydrophobicity analysis (see, e.g., Kyte and Doolittle, 1982, or U.S. Pat. No. 4,554,101). Hydrophobicity analysis assigns average hydrophilicity values to each amino acid residue, and from these values average hydrophilicities can be calculated and regions of greatest hydrophilicity determined. Using one or more of these methods, regions of predicted antigenicity may be derived from the amino acid sequence of the disclosed P13 polypeptides. Proposed epitopic regions from the disclosed P13 antigens include the sequences corresponding to amino acid residues 7-15, 21-24, 29-35, 83-92, 126-135 and 162-167 in SEQ ID NO: 19; amino acid residues 7-14, 20-23, 28-35, 82-89, 125-134 and 162-166 in SEQ ID NO: 21; and amino acid 35 residues 6-14, 18-21, 27-34, 79-92, 125-133 and 161-165 in SEQ ID NO: 23.
Antigenic epitopes can also be determined using different experimental procedures known to the skilled person. For example, the DNA encoding the P13 polypeptides can be digested with restriction enzymes in such a manner that DNA fragments encoding specific parts of the P13 polypeptide are obtained. These DNA fragments can be expressed in a suitable expression system. The P13 polypeptide fragments obtained can be analysed with monoclonal or polyclonal antibodies obtained by immunisation with the full-length form of the P13 polypeptide or fragments thereof, or with Lyme disease patient sera to obtain information about immunogenicity and the presence of epitopes. Fragments found to be positive in such a simple screening assay where they react specifically with e.g. polyclonals raised against P13 are thus suitable candidates for a multitude of the applications where full-length P13 would also be suitable.
A similar approach can be used where random mutations are introduced in the nucleotide sequence encoding P13, and only the expression products which retain a suitable reactivity with e.g. P13 positive polyclonal antibodies will be used as candidates for further applications.
Peptide fragments for the identification of epitopes can also be obtained by synthetic methods. Suitable peptides can be synthetised based on the amino acid sequence of the P13 polypeptides, e.g. peptides with amino acid sequences identical to consecutive fragments from the N-terminus to the C-terminus of the P13 polypeptide can be synthetised on solid-phase media. Such custom-made peptide libraries can be obtained from commercial sources.
Finally, another way of simply identifying epitopes is to digest a polypeptide antigen with a known amino acid sequence with endo- and exopeptidases. The obtained fragments are tested against antibodies directed against the whole polypeptide, and by way of deduction, the precise location of the linear epitopes can be determined. A variation of this method involves the recombinant production of subfragments (cf. the above) of the full-length polypeptide followed by the same test procedure.
It is contemplated that the antigens and immunogens of the invention will be useful in providing the basis for one or more assays to detect antibodies against Bb. Previous assays have used whole Bb as the antigen. Sera from normal individuals not exposed to Bb often contain antibodies that react with Bb antigens, in particular antigens that have epitopes in common with other bacteria. It is necessary to adjust assay conditions or the diagnostic threshold of reactivity to avoid false positive reactions due to these cross-reactive antibodies in normal sera. These adjustments may in turn decrease the sensitivity of the assay and lead to false negative reactions, particularly in the early stages of Bb infection. Assays using the disclosed P13 proteins or antigenic polypeptides thereof are expected to give superior results both in terms of sensitivity and selectivity when compared to assays that use whole Bb or even purified flagella in either an indirect ELISA or an antibody capture ELISA format. Western immunoblots based on reactions with such antigens (whole Bb, flagella and the like) have been difficult to interpret due to the presence of antibodies in sera from unexposed individuals. These antibodies cross-react with Bb antigens, most particularly the 41 kDa flagellin and the 60 kDa common antigen protein. Generally, assays which use whole organisms or purified flagella tend to contain antigens with epitopes that will cross-react with other bacterial antigens. For example, the N and C terminal regions of the Bb flagellin possess 52-55% sequence identity with the Salmonella typhimurium and Bacillus subtilis sequences (Wallich et al., 1990), exemplifying the highly conserved nature of flagellin structure. The 60 kDa Bb protein is likewise 58% homologous with the E. coli protein (Shanafelt et al., 1991). Such cross-reactivity is not likely with the disclosed P13 antigens, which are apparently unique to Bb.
It is further anticipated that recombinantly derived P13 Bb proteins will be particularly preferred for detecting Bb infections. Unexposed individuals should have a low reactivity to one or more epitopes of the P13 proteins thereby making it possible to use lower dilutions of serum and increase sensitivity. Using a combination of more than one of these unique antigens may also enhance sensitivity without sacrificing specificity.
Preferred immunoassays are contemplated as including various types of enzyme linked immunoassays (ELISAs), immunoblot techniques, and the like, known in the art. However, it readily appreciated that utility is not limited to such assays, and useful embodiments include RIAs and other non-enzyme linked antibody binding assays or procedures.
Yet another aspect of the invention is a method of detecting Bb nucleic acid in a sample. The presence of Bb nucleic acid in the sample may be indicated by the presence of the polypeptide products which it encodes. The method therefore includes detecting the presence of at least a portion of any of the polypeptides herein disclosed. Suitable detection methods include, for example, immunodetection reagents, PCR amplification, and hybridization.
Yet another aspect of the invention includes one or more primers capable of priming amplification of the disclosed DNA of SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22. Such primers are readily generated taking into account the base sequence of the DNA segment of SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22, the disclosed DNA, or deriving a base sequence from the amino acid sequence of a purified polypeptide encoded by the DNA. Primers are analogous to hybridization probes, but are generally relatively short DNA segments, usually about 7-20 nucleotides.
Methods of diagnosing Lyme disease are also included in the invention. In one embodiment, an antibody-based method includes obtaining a sample from a patient suspected of having Lyme disease, exposing that sample to one or more epitopes of the Bb protein which is encoded by the DNA disclosed, and finally determining a reactivity of the antibody with one or more epitopes of a Bb protein that may be in the sample. The reactivity measured is indicative of the presence of Lyme disease. Typical samples obtainable from a patient include human serum, plasma, whole blood, cerebrospinal fluid, seminal or vaginal fluids, exudates and the like.
Several variations of antibody-based methods are contemplated for development; for example, an indirect ELISA using the P13 proteins or other Bb proteins as an antigen. The P13 proteins may be produced in large quantities by recombinant DNA vectors already disclosed and purified. Optimum concentration of the antigen could be determined by checker board titration and diagnostic potential of the P13 proteins assay examined further by testing serum from mice at different stages of infection and infected with different strains of Bb. These results could indicate the relative time course for serum conversion for each of the assays and would also show whether infection with different strains causes variation in anti-P13 protein titers.
Likewise, reactive epitopes of the P13 polypeptides are contemplated as useful either as antigens in an ELISA assay or to inhibit the reaction of antibodies toward intact P13 proteins bound to a well. Epitopic peptides could be generated by recombinant DNA techniques previously disclosed or by synthesis of peptides from individual amino acids. In either case, reaction with a given peptide would indicate presence of antibodies directed against one or more epitopes. In addition to its diagnostic potential, this method is seen as being particularly effective in characterising monoclonal antibodies against the P13 proteins and other virulence associated proteins.
In a further aspect, the present invention concerns a kit for the detection of Bb antigens, the kit including a protein or peptide which includes an epitope thereof, together with means for detecting a specific immunoreaction between an antibody and its corresponding antigen. Examples of suitable means include labels attached directly to the antigen or antibody, a secondary antibody having specificity for human Ig, or protein A or protein G. Alternatively, avidin-biotin mediated Staphylococcus aureus binding could be used. For example, the monoclonal antibody may be biotinylated so as to react with avidin complexed with an enzyme or a fluorescent compound.
A particular kit embodiment of the invention concerns detection of antibodies against the described Bb P13 antigens, epitopes thereof as represented by portions of the amino acid sequences, or closely related proteins or peptides, such as epitopes associated with other virulence-associated proteins detected by comparison of low-passage, virulent and high-passage, avirulent strains of Bb. The antigen for the kit(s) consists of the Bb P13 proteins or portions thereof produced by a recombinant DNA vector in E. coli or another bacterial or non-bacterial host. Alternatively, the antigen may be purified directly from Bb or manufactured as a synthetic peptide. Samples for the assays may be body fluids or other tissue samples from humans or animals. The presence of reactive antibodies in the samples may be demonstrated by antibody binding to antigen followed by detection of the antibody-antigen complex by any of a number of methods, including ELISA, TRIFMA (time-resolved immunofluorometric assay), RIA, fluorescence, agglutination or precipitation reactions, nephelometry, or any of these assays using avidin-biotin reactions. The degree of reactivity may be assessed by comparison to control samples, and the degree of reactivity used as a measure of present or past infection with Bb. The assay(s) could also be used to monitor reactivity during the course of Lyme disease, e.g., to determine the efficiency of therapy.
In still further embodiments, the invention contemplates a kit for the detection of Bb nucleic acids in the sample, wherein the kit includes one or more nucleic acid probes specific for the P13 genes, together with means for detecting a specific hybridization between such a probe and Bb nucleic acid, such as an associated label.